I will use confocal microscopy and volume rendering to study the cell biology of fiber cell formation in normal and cataractous lenses. I have developed a lens slice preparation, which allows cells lying in the midsagittal plane of the lens to be imaged at high resolution. I will employ the lens slice to study organelle loss during fiber cell maturation, using fluorescent vital-dyes to follow the fate of organelles in living lens tissue. Preliminary studies on the fate of mitochondria and nuclei indicate that these organelles are lost from embryonic fiber cells in an abrupt, coordinated fashion in a restricted domain of the deep cortex. I will investigate the mechanism of mitochondrial degradation and the role of reduced 02 tension in this process. Isolated mitochondria will be used to study the degradative process in vitro. I will extend these studies to other organelles, including the Golgi apparatus, endoplasmic reticulum (ER) and the cytoskeleton. I will compare organelle distribution in adult lenses from several species and two cataract models. Lens fiber cells are among the longest in the body. I have preliminary data that demonstrate that the ER is specifically located near the posterior tips of the fibers. This localization may reflect a requirement for the ER to be located close to the site of protein secretion. I will determine whether collagen mRNAs are also localized at the posterior tips of the fibers, where collagen is secreted during capsule formation. Another expected consequence of the elongated fiber cell morphology is the presence of intracellular transport system. Using time-lapse confocal microscopy, I obtained preliminary data indicating the presence of such mechanisms in lens fiber cells. The kinetics of transport will be characterized in lens slices. Studies will focus on the role of the cytoskeleton in this process. Throughout this project, computerized volume reconstruction (volume rendering) will be used to visualize the inter-relationship of cellular elements in 3-D. This will be particularly important in the final phase of the proposal, where dynamic 3-D growth processes will be imaged and quantitated. The formation of the lens suture is crucial to lens function and is often disturbed in cataracts. I will use confocal microscopy to observe the formation of the suture in real time in living, organ cultured embryonic lenses. This should reveal critical morphological determinants of suture formation. Cell shape and volume are inextricably linked during lens growth. I will test the hypothesis that the cross-sectional shapes of lens fiber cells are determined by volume increases in the epithelial cells from which they are derived. These studies will require careful 3-D reconstruction of embryonic lenses and dynamic imaging of cells that have been stimulated to elongate.